CoA is a substance which functions as an acyl carrier/acyl activator in all biological species. For example, in addition to the fact that acetyl CoA is a key substance for important biological metabolism of fatty acid, glucose, etc. via a citric acid cycle, some kinds of acyl CoA derivatives play an important role in biosynthesis of cholesterol and fatty acid as well. CoA is essential as an auxiliary factor (coenzyme) for an enzymatically catalyzing reaction (CoA enzyme) concerning such a metabolism, is a substance which is unable to be substituted with others and is represented by the following formula.
(In the Formula, ACY is an Acyl Group.)
In CoA enzyme, there are various CoA enzymes depending upon the structure and the substrate (a compound into which an acyl group is to be introduced) of an acyl group to be transferred. Up to now, many attempts have been made for the production of a substance using various kinds of CoA enzymes and, for example, there are examples for the production of antibiotics and drugs, various chemical substances utilizing a polyketide synthesis route, amino acids, polyhydroxy acids, etc.
In those methods, equimolar acyl CoA is consumed as an acyl group receptor. Accordingly, it is required that the necessary acyl CoA is produced at a low cost.
In any of the above-mentioned methods, acyl CoA in vivo which is fermentationally produced by fermentation or acyl CoA which is produced separately from the production system is used. When acyl CoA in vivo fermentationally produced is used, only a specific acyl CoA produced in vivo such as acetyl CoA or malonyl CoA is able to be utilized. In order to solve such a problem, there has been reported an art where an enzymatic ester exchange is carried out between acetyl CoA and various fatty acids to produce acyl CoA in vitro. The method where acyl CoA which is separately produced from the production system is used has a multiplicity of uses and is a method which has been commonly used but the acyl CoA which is produced as such is very expensive and it is still necessary to use equimolar amount when used for a transfer reaction of an acyl group.
With regard to a production process of acyl CoA, a chemical synthetic method using an acyl chloride, a chemical synthetic method using an acid anhydride, a chemical synthetic method using a mixed acid anhydride with ethyl chlorocarbonate, a chemical synthetic method by a thioester exchange (Z. Naturforsch. 29C, 469-474 (1974); Z. Naturforsch. 30C, 352-358 (1975); J. Am. Chem. Soc., 1953, 75, 2520; J. Biol. Chem., 1985, 260, 13181) and many other chemical synthetic methods have been generally used. However, in many of the chemical synthetic methods, selectivity to thiol group is low in general and there is a problem that the yield is lowered by a non-selective acylation reaction. Although these production processes have been used even today, they are merely used for a laboratory production of acyl CoA.
In order to overcome the weak points of the chemical synthetic method, an enzymatic production process of acyl CoA has been also studied vigorously. Thus, a method using an acetyl CoA synthetic enzyme, a method using a fatty acid CoA synthetic enzyme, etc. have been reported (Appl. Microbiol. Biotechnol., 1994, 40, 699-709). However, in those enzymatic reaction methods, it is very difficult to obtain the enzyme which serves as a catalyst in a necessary amount.
With regard to a production process using an enzyme, a study concerning a coupling method in cooperation with a CoA enzyme reaction using it as an acyl CoA reproduction system has been reported. That is, the acyl CoA consumed by an acyl group transfer reaction is reproduced by an enzymatic reaction and used for the reaction again and there are a coupling method using phosphotransacetylase, a coupling method using carnitine acetyltransferase, a coupling method using an acetyl CoA synthetic enzyme, a coupling method using an α-ketoglutaric acid dehydrogenase, etc. These methods have a high selectivity to thiol group and particularly an acetyl CoA synthetic enzyme has wide substrate selectivity and is useful because various kinds of acyl CoAs are able to be generated.
However, these methods reproduce an acyl CoA by enzyme and each of them has problems including that such an enzymatic reproduction system has a slow reaction rate, enzyme is unstable, ATP and a relatively expensive auxiliary component are necessary for the reaction and no reaction is possible in CoA of high concentrations. Thus, unless the price of the aimed product is considerably high, they are generally said to be unable to be industrial production methods in terms of cost if an acyl CoA is used for less than 10,000 times. Consequently, the above-mentioned methods are not satisfactory as industrial production methods. Although there is an attempt to reproduce an acetyl CoA utilizing a non-enzymatic reaction using an N-acetyl substance of dimethylaminopyridine (Bioorganic Chem., 1990, 18, 131-135), it is a bilayer system using a large quantity of an organic solvent whereby there is a problem in view of purification of the product and the method is not suitable for an industrial production.
As mentioned above, an acyl CoA reproduction system which is satisfactory for enabling the utilization of a CoA enzyme as an industrial production method has not been known up to now.
Sphingolipid is a lipid derived from a sphingoid base such as sphingosine and is present in cell membranes of animals, plants and microbes. Although a precise function of human sphingolipid has not been known yet, a group of such compounds participates in electric signal transmittance in a nervous system and stabilization of cell membranes. Sphingoglycolipid has a function in immune system and it has been shown that a specific sphingoglycolipid functions as a receptor for bacterial toxin and also probably as a receptor for microbes and viruses.
Ceramide is a specific group of sphingolipid containing sphingosine, dihydrosphingosine or phytosphingosine as a base. Ceramide is a main lipid component of horny layer which is an upper layer of the skin and has an important barrier function. It has been known that a topical application of a composition containing a sphingolipid such as ceramide improves, for example, a barrier function and a moisture-retaining characteristic of the skin (Curatolo, Pharm. Res., 4: 271-277 (1987); Kerscher, et al., Eur. J. Dermatol., 1:39-43 (1991)).
It has been known that a sphingoid base per se inhibits the activity of protein kinase C which is an important enzyme in a signaling pathway and accordingly that it mediates several physiological actions. Moreover, a sphingoid base is contained in cosmetic compositions or in dermatological compositions due to its anti-inflammatory activity and antibacterial activity.
At present, heterosphingolipid preparations for cosmetics are mostly extracted from animal sources. However, that is a method which is relatively expensive in an industrial scale and a public concern is increasing for novel material sources for pure and structurally specified sphingolipid which is available from other supplying sources than animal tissues because of, for example, a latency of bovine spongiform encephalopathy (BSE).
It has been found that microbe such as Pichia ciferrii yeast produces sphingolipid, sphingosine, phytosphingosine and/or derivatives thereof (Wickerham and Stodola, J. Bacteriol., 80:484-491 (1960)). Such a microbe provides supplying source for sphingolipid per se and supplying source for starting material for production of other commercially valuable compounds and gives an practically applicable substitute to the use of animal supplying source for those compounds. However, in the production by microbes, improvement in productivity is difficult because of toxicity of a sphingoid base to microbe cells (Pinto, et al., J. Bacteriol., 174:2565-2574 (1992); Bibel, et al., J. Invest. Dermatol., 93:269-273 (1992)) and there has been a brisk demand for providing more efficient production process.
In addition, as a result of increasing consciousness to environmental issues in recent years, there is much more interest in biodegradable macromolecules being friendly to environment than in synthetic macromolecules which have occupied the main stream.
Polyhydroxy alkanoate (hereinafter, may be abbreviated as PHA) which is one of biodegradable macromolecules is a polyester being usually produced by a fermentation production of microbes and receiving public attention due to its high biodegradability and 90 or more kinds have been known (FEMS Microbiol. Lett., 1995, 128, 219-228). Among them, research and development have been promoted for poly(3-hydroxybutyrate) (hereinafter, may be abbreviated as PHB), poly(3-hydroxyvalerate) (hereinafter, may be abbreviated as PHV) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (hereinafter, may be abbreviated as PHB-co-PHV) due to ease of produce and good characteristics (Japanese Laid-Open Patent Publication No. 57-150393 (U.S. Pat. No. 4,393,167), Japanese Laid-Open Patent Publication No. 59-220192 (European Patent Laid-Open No. 0114086), Japanese Laid-Open Patent Publication No. 63-226291 (European Patent Laid-Open No. 0274151) and Japanese Laid-Open Patent Publication No. 63-269989). However, there are many problems in PHA that, the productivity is low in the production by fermentation of microbe in order to accumulate PHA in microbe cells and that, in addition, it takes much cost for purification by crushing microbes and extracting PHA.
Since then, analysis of the mechanism of fermentation production has proceeded, which increased accumulated concentration of PHA into the microbe cells significantly, and also analysis of mechanism of accumulated state of PHA into microbe cells has proceeded, which lowered cost for extraction and purification of PHA from microbes whereby actual production of PHA using microbes has started.
In addition, since it has been in the meanwhile clarified that there are varieties of microbes which produce PHA, research and development of PHA other than PHB, PHV and PHB-co-PHV has made substantial progress and research and development of copolymers for improving the physical property have been also carried out (Japanese Laid-Open Patent Publication Nos. 63-269989, 64-048821, 01-156320, 01-222788 and 05-093049).
However, since a production process of PHA by fermentation production of microbe proceeds via a complicated biometabolic path, the desired PHA is not always produced and, moreover, variation of PHA is limited as well. Further, depending upon a method for controlling the fermentation production, a desired homopolymer is not produced but a copolymer is formed and, reversely, a homogeneous copolymer in a desired polymerization ratio is not always produced in a production of copolymer (FEMS Microbiol. Rev., 1992, 103, 207-214). In addition, in a purification step, since a desired PHA is taken out from microbe cells containing many kinds of compounds, there is a limitation in improving purity in an industrial production. As such, production of PHA by fermentation of microbes has various problems.
On the other hand, by a genetic recombination technique which has quickly progressed in recent years, gene of polyhydroxyalkanoate synthase (PHAS) which is an enzyme copolymerizing PHA was isolated and, by enhancing its expression, improvement of production of PHA has been also attempted (Japanese Laid-Open Patent Publication Nos. 07-265065, 10-108682 and 2001-516574 (WO 99/14313)).
Further, it is now also possible to separate and purify PHAS in large quantities using a genetic recombination technique and a method for polymerization of PHB in vitro without using a microbe fermentation has been developed whereby homogeneous and highly pure PHB is able to be produced (Proc. Natl. Acad. Sci., 1995, 92, 6279-6283,; Int. Symp. Bacterial Polyhydroxyalkanoates, 1996, 28-35; Eur. J. Biochem., 1994, 226, 71-80; Appl. Microbiol. Biotechnol., 1998, 49, 258-266; Macromolecules, 2000, 33, 229-231).
After that, it has been shown that PHA other than PHB is also able to be synthesized by the similar in vitro polymerization method and there is no limitation on variation of PHA which has been unable to be achieved by a microbe fermentation method whereby it is suggested that variation of PHA is significantly expanded (Biomacromolecules, 2000, 1, 433-439; Appl. Microbiol. Biotechnol., 2001, 56, 131-136; Macromolecules, 2001, 34, 6889-6894). In that method, it is also possible to synthesize copolymers in addition to homopolymers.
However, acyl CoA is to be used as a starting substance for the polymerization in an in vitro polymerization method, but, as mentioned above, there are various problems for the synthesis of acyl CoA.
Accordingly, there has been a demand for suppressing the amount of an acyl CoA used very small and also for developing a production process of macromolecular compounds where other compound which is easily synthesized industrially is used as a starting substance.
On the other hand, in an in vitro polymerization method, acyl CoA is used as a substrate for enzyme and the enzyme reacts whereupon PHA is polymerized and, at the same time, liberated CoA is discharged into the reaction system (refer to the following formula).

(In the formula, R0 is an organic group wherein R0—SH is CoA; R1 is any alkylene; and n is an integer corresponding to degree of polymerization.)
As such, each time when a reaction of acyl group transfer from acyl CoA takes place, one repeating unit is added whereupon one molecule of CoA is released.
In an in vitro polymerization method, this CoA remains in a reaction system in its free state just to be accumulated therein and the yield of a macromolecular polymerization reaction does not exceed the equivalent amount of the acyl CoA which is put into the reaction system. Therefore, productivity of PHA is very low and cost of PHA manufactured by an in vitro polymerization method is nothing but quite expensive. As the polymerization proceeds further, CoA concentration in the reaction system increases whereby there are concerns about an inhibition effect to the enzymatic reaction as well.
Incidentally, as an effective utilization method of CoA which is present in a high concentration in the reaction system in a free state, its reproduction is attempted as well (FEMS Microbiology Letters, 1998, 168, 319-324). That is, acetic acid, acetyl CoA synthetase and ATP are made coexisted in a polymerization enzymatic reaction solution whereby CoA which is liberated after the polymerization reaction is converted to acetyl CoA and, in addition, propionyl CoA transferase and 3-hydroxybutyrate are also made coexisted to give 3-hydroxybutyrate CoA which is a substrate for the polymerization catalyst. In that method however, as many as three kinds of enzymes which are very difficult to purify are used and, further, quite expensive ATP is also necessary whereby it is very difficult to apply it to an industrial production process.
As such, in an in vitro polymerization method, it is necessary to use an acyl CoA as a reaction substrate and, since acyl CoA is very expensive, it is quite difficult to lower the production cost of PHA when acyl CoA is used as a reaction substrate for an industrial production of PHA. Moreover, in a reproduction of CoA to acyl CoA, many kinds of enzymes which are difficult to obtain are necessary and, in addition, an expensive compound such as ATP is necessary. Further, in a production process of PHA using living organism or, particularly, microbe, variation of PHA is limited and, furthermore, there is a high possibility that copolymer is polymerized due to metabolism in living body whereby it is difficult to produce a desired PHA only. In view of the above, there has been a demand for developing a production process which enables greater variation of PHA, and can lower the production cost of PHA by using a compound which is easily able to be synthesized in its production as a starting substance.